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Ramanuj DasGupta, Horace Rhee and Elaine Fuchs

Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021

Address correspondence to Elaine Fuchs, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave., Box 300, New York, NY 10021. Tel.: (212) 327-7953. Fax: (212) 327-7954. E-mail: Fuchs{at}rockefeller.edu

	Abstract

Top Abstract Introduction Results Discussion Materials and methods References

 
Wnt signaling orchestrates morphogenetic processes in which changes in gene expression are associated with dramatic changes in cell organization within developing tissue/organss. Upon signaling, excess ß-catenin not utilized at cell–cell junctions becomes stabilized, where it can provide the transcriptional activating domain for Lef/Tcf DNA binding proteins. In skin epithelium, forced stabilization of ß-catenin in epidermis promotes hair follicle morphogenesis, whereas conditional removal of ß-catenin in hair progenitor cells specifies an epidermal fate. We now report that a single protein, a stabilized version of ß-catenin lacking the COOH-terminal transactivation domain, acts in epidermis to promote hair fates and in hair cells to promote epidermal fate. This reveals fundamental differences in ways that epidermal and hair cells naturally respond to ß-catenin signaling. In exploring the phenotype, we uncovered mechanistic insights into the complexities of Lef1/Tcf/ß-catenin signaling. Importantly, how a cell will respond to the transgene product, where it will be localized, and whether it can lead to activation of endogenous ß-catenin/Tcf/Lef complexes is specifically tailored to skin stem cells, their particular lineage and their relative stage of differentiation. Finally, by varying the level of ß-catenin signaling during a cell fate program, the skin cell appears to be pliable, switching fates multiple times.

Key Words: Wnt signaling; epidermis; hair follicle; ß-catenin; morphogenesis

	Introduction

Top Abstract Introduction Results Discussion Materials and methods References

 
Cells able to receive and transduce a Wnt signal respond by stabilizing any excess ß-catenin not used in cell–cell junction formation and recruiting it to act as a transcriptional cofactor for the activation of genes regulated by the Lef1/Tcf family of DNA binding proteins (Huelsken and Birchmeier, 2001; Brantjes et al., 2002). In cells that make cell–cell junctions, ß-catenin is naturally stabilized through its interaction with the adherens junction components, E-cadherin and -catenin (Jou et al., 1995; Aberle et al., 1996; Gottardi and Gumbiner, 2001; Nagafuchi, 2001). Excess cytoplasmic ß-catenin is phosphorylated at its NH₂-terminal serine-threonine residues by the GSK3ß kinase, and targeted for proteosome-mediated degradation by a complex that includes the tumor-suppressor gene product adenomatous polyposis coli (APC)^* (Behrens et al., 1998; Henderson, 2000; Reinacher-Schick and Gumbiner, 2001; van Noort et al., 2002). When a cell's Wnt surface receptors are stimulated, they can activate a signal transduction cascade that culminates in the inhibition of GSK3ß, leading to the accumulation of ß-catenin. An artificial promoter Tcf-optimal-promoter (TOP) containing multimerized Lef1/Tcf binding sites, has been used in conjunction with various reporter genes to identify Wnt-responsive cells (Molenaar et al., 1996; DasGupta and Fuchs, 1999; for review see Brantjes et al., 2002).

Recent studies have implicated members of the Wnt signaling pathway in the epithelial–mesenchymal exchanges that specify morphogenesis of the hair follicle (for review see Hardy, 1992; Fuchs et al., 2001). In mouse, TOP-ßgalactosidase (TOPGAL) activity was detected in embryonic skin at sites of hair follicle formation, and postnatally, TOPGAL was active in the stem cell compartment (bulge) at the beginning of each hair cycle and also in the precursor cells that differentiate into hairs (DasGupta and Fuchs, 1999). The studies of Kishimoto et al. (2000) suggest that Wnts maintain the inductive ability of mesenchyme to stimulate hair cell fate at the start of each cycle (Kishimoto et al., 2000).

Evidence that Wnt signaling is functionally important for hair follicle morphogenesis stems from transgenic mice engineered to express a constitutively stable and active form of ß-catenin under the control of an epidermal keratin promoter (K14) (Gat et al., 1998). In epidermis, its expression spontaneously induced aberrant hair follicles throughout the interfollicular epidermis between the preexisting follicles (Gat et al., 1998). Similarly, in chick embryos, what appeared to be sustained activation of ß-catenin led to excessive feather and scale morphogenesis (Noramly et al., 1999; Widelitz et al., 2000). Conversely, ablation of Lef1 gene expression in mouse skin or ß-catenin expression in K14-expressing cells of mouse skin impairs hair follicle morphogenesis (van Genderen et al., 1994; Huelsken et al., 2001). Based on these findings, Wnt signaling seems to coax skin stem cells along a hair (epidermal appendage) cell fate. Consistent with this notion is the finding that the hair-specific keratin promoters are bona fide target genes of Lef/ß-catenin signaling (Zhou et al., 1995; Merrill et al., 2001). Additionally, concomitant with the development of a hair placode is an upregulation in transcription of both Lef1 and ß-catenin genes (Zhou et al., 1995; Noramly et al., 1999; Widelitz et al., 2000; Huelsken et al., 2001).

Although Wnt signaling seems to promote hair cell fates, an absence of or interference with Wnt signaling seems to favor epidermal and sebocyte cell fates. Thus, a K14-conditional loss of function mutation in the ß-catenin gene locus resulted in a blockage of embryonic hair development if lost early and formation of epidermal-like cysts in place of hair follicles if lost later (Huelsken et al., 2001). In addition, transgenic mice expressing K14-NLef1, unable to bind ß-catenin, yielded epidermal and sebocyte differentiation at sites where postnatal hair cycling was expected (Merrill et al., 2001; Niemann et al., 2002).

The picture emerging from these studies suggests that setting the levels of ß-catenin plays a crucial role in deciding if stem cells or their progeny will contribute to structures below the bulge (hair cell types) or above the bulge (sebaceous gland, upper outer root sheath [ORS], and epidermis). These findings have prompted us to wonder whether epidermal and hair follicle cells are truly equivalent with respect to their internal ability to translate a Wnt signal, or whether there might be important internal differences that might be overridden and obscured by overexpressing or ablating ß-catenin in mice. If, as recent evidence implies, stem cell progeny migrate from the bulge and specify cell fates in different environments (Taylor et al., 2000; Oshima et al., 2001), do they maintain equivalency with respect to their ability to respond to a stabilized ß-catenin signal, or does this chemistry change? Is it simply differences in Wnt/Wnt receptors, or are there distinct differences in the way hair and epidermal cells respond to increased ß-catenin expression?

Placed in a broader context of cell lineage determination, the heart of this problem focuses on the extent to which the levels of ß-catenin's varied interacting partners might differ in cells and how this impacts on the manner in which the cell responds to a Wnt signal and/or stabilized ß-catenin. The skin becomes an interesting model system to tackle this problem, because not only is the hair versus epidermal fate manipulated by overexpression or underexpression of ß-catenin, but in addition, at least some of ß-catenin's associates, including Lef1/Tcf levels (Zhou et al., 1995; DasGupta and Fuchs, 1999; Merrill et al., 2001) and intercellular junction proteins (Nanba et al., 2000) are known to be differentially expressed in these two cell types.

To determine the extent to which hair and epidermal cells might differ in their internal ability to process and respond to a given level of ß-catenin, we sought a strategy for manipulating ß-catenin signaling in a way in which we would expect to see the same phenotypic outcomes in mouse epidermal and hair cells if they responded equivalently, but different outcomes if they harbored distinctly different means of receiving the same signal. Rather than stabilizing ß-catenin and seeing hair follicle morphogenesis in epidermis as well as hair follicles (Gat et al., 1998), or ablating ß-catenin and seeing epidermal differentiation in hair follicles as well as in epidermis (Huelsken et al., 2001), we searched for conditions in which epidermal cells responded in one way and hair cells in another. We settled on engineering mice to express a COOH terminally truncated form of the constitutively stabilized version of ß-catenin that we had used earlier in generating our super-furry mice (Gat et al., 1998).

The COOH terminus of ß-catenin is required for transactivation, and it binds the transcriptional activator p300/CBP as well as the chromatin remodeling factor Brg-1 (Molenaar et al., 1996; Orsulic and Peifer, 1996; van de Wetering et al., 1997; Huber et al., 1997; Hsu et al., 1998; Hecht et al., 1999, 2000; Barker et al., 2001). Indeed, ß-catenin mutants lacking the COOH terminus can be deficient in signaling not only in vitro, but also in vivo (Orsulic and Peifer, 1996; Cox et al., 1999; Hecht et al., 1999; Vleminckx et al., 1999; Barker et al., 2001; Tutter et al., 2001). This said, prior studies on ß-catenin signaling in Xenopus and in Drosophila had demonstrated that overexpression of membrane-tethered or NH₂/COOH terminally truncated forms of ß-catenin could potentiate signaling depending on its interacting partners within the cell. This led to the hypothesis that these forms of ß-catenin might act by displacing endogenous ß-catenin from intercellular junctions, freeing it to be utilized in transactivation (Miller and Moon, 1997; Cox et al., 1999).

By expressing a single protein with potentially dominant negative and dominant positive character, we find that different cells within the skin behave unidirectionally towards this protein. However, strikingly and ironically, whereas the follicle stem cells and their progeny respond in a dominant negative fashion to the transgene product, the epidermal cells respond in a dominant positive fashion. Moreover, we show that by changing transgene expression midstream in the process, the process becomes reversible, and cells can revert back to their original lineage. Our findings have important and broad implications for how cells respond to and translate Wnt/ß-catenin signals and how cell type specificity and cell fate commitment is achieved.

	Results

Top Abstract Introduction Results Discussion Materials and methods References

 
K14-NCßcatenin interferes with the transactivation of target genes in keratinocytes in vitro
To engineer K14-NCßcatenin encoding a stable form of ß-catenin that lacks Tcf/Lef1-mediated signaling activity, we used the K14-N87ßcatenin transgene (Gat et al., 1998) and removed sequences encoding the COOH-terminal 98 amino acid residues of ß-catenin (Fig. 1 A). We also added a COOH-terminal HA-epitope tag so we could easily monitor transgene expression in vitro and in vivo. Addition of this epitope was previously found to yield functional Nßcatenin (Gat et al., 1998; unpublished data). To verify that the transgene encoded a protein of the expected size, we transiently transfected K14-NCßcatenin into mouse keratinocytes. 48 h after transfection, cells were processed for immunoblot analysis using a rat anti-HA monoclonal antibody. Only cells transfected with K14-NCßcatenin showed a band of the correct size (66.5 kD), whereas mock-transfected cells displayed no expression (Fig. 1 B). The blot was probed with an anti-tubulin antibody to verify that the protein loadings were equivalent.

View larger version (15K): [in this window] [in a new window]   Figure 1. The K14-NCßcatenin construct and in vitro transfection assays. (A) The K14-NCßcatenin construct starting with amino acid 88 at the NH2 terminus of human ß-catenin and ending at amino acid 682. The K14 expression construct comprises the K14 promoter at the very 5'-end. It also includes the ß-globin intron, the COOH-terminal HA tag, and pA, the poly-adenylation signal, which is at the 3' end of the construct. (B) Western blot using the anti-HA antibody on protein extracts derived from control K14 empty vector transfected and K14-NCßcatenin–transfected mouse keratinocytes. Note that a 66.5-kD band exists only in extracts from cells transfected with NCßcatenin. (C) Transient transfection assays with TOPGAL reporter. Mouse keratinocyte line, RD1, was transfected with pTOPGAL, pCMV-luciferase (as an internal control gene) and equimolar amounts of plasmids K14-N87ßcat, K14-NCß-cat, pK14-Lef1, or empty expression vector, as indicated (see Gat et al., 1998, for method). 36–48 h later, cells were lysed and protein extracts were assayed for ß-galactosidase activity (test) and luciferase (to correct for transfection efficiency). Normalized activities of TOPGAL (black bars) represent an average of three experiments, with variations shown by error bars. FOPGAL (white bars) had no activity. Whereas Lef1 and Nßcat super-activated the TOPGAL reporter, Lef1 and NCß-cat failed to do so. (D) K14–NCßcat acts like a dominant negative; same as C, but with equimolar amounts of K14-Nßcat and K14–NCßcat added together with Lef1, which blocked the activation mediated by Lef1 and Nßcat alone.

Similar to that seen previously for TOPFLASH (Gat et al., 1998), Nßcatenin and Lef1 on their own each had only a modest effect on TOPGAL reporter activity, but together, they strongly transactivated (Fig. 1 C). These effects were dependent on the presence of functional Lef1 binding sites, as evidenced by the lack of activation with FOPGAL mutant in the Lef1 binding sites (Fig. 1 C). In contrast, Lef1 and NCßcatenin together exhibited only slightly higher levels than Lef1 alone, although the activity was consistently above background (Fig. 1 C). Thus, despite the fact that NCßcatenin lacks the transactivating domain, some activation was still observed when it was coexpressed with Lef1 in keratinocytes. We will return to this point again later.

When equal amounts of Nßcatenin and NCßcatenin were expressed together with Lef1, NCßcatenin suppressed the activation of the TOPGAL reporter by twofold (Fig. 1 D). Similar inhibitory effects were observed with Tcf3, although because Tcf3 normally functions as a repressor, this was more difficult to demonstrate (unpublished data). The dominant negative effects of NCßcatenin were expected, given that NCßcatenin still contained the armadillo repeats, able to interact with the Tcf/Lef1 DNA binding proteins (Huber et al., 1997; Graham et al., 2000; Tutter et al., 2001). Thus, irrespective of which Lef1/Tcf family member NCßcatenin was combined with, NCßcatenin impaired the transactivation potential conferred to by stabilized ß-catenin.

Expression of NCßcatenin in the epidermis, follicle ORS, and stem cell compartment of transgenic mice
Given the ability of NCßcatenin to act in a dominant negative fashion in interfering with the activity of functional Lef1/ßcatenin complexes in keratinocytes in vitro, we then turned towards using this transgene to assess the consequences of diminished Wnt signaling on hair organogenesis and postnatal cycling. For purposes of comparison to the previously generated K14-Nßcatenin and K14-Cre conditional ß-catenin–null mice, we used the K14 promoter to drive the expression of NCßcatenin in the dividing cells of the epidermis, the ORS, and the stem cell compartment of hair follicles.

K14-NCßcatenin mice were generated in the CD-1 strain, and PCR of tail DNAs confirmed the identity of mice that carried the transgene. Expression of NCßcatenin protein was confirmed by immunoblot analysis of proteins isolated from whole newborn skin of transgenic and control littermate animals, as well as from primary keratinocytes derived K14-NCßcatenin–expressing mice. In transgenic keratinocytes in vivo and in vitro, the transgenic protein of 66.5 kD was detected by the anti-HA antibody (Fig. 2 A). Immunofluorescence analysis using the HA antibody on frozen skin sections confirmed the expression of the transgene specifically in the basal epidermal layer (Fig. 2 B) and the follicle ORS and the bulge (Bu) (Fig. 2, B' and C, schematic of follicle). Two independently derived mouse lines behaved similarly in this and all subsequent analyses, thereby attributing the observed effects to transgene expression and not chromosomal integration site.

View larger version (31K): [in this window] [in a new window]   Figure 2. Stable expression of HA-tagged NCßcatenin transgene in skin and primary keratinocytes. (A) Immunoblot analysis of K14-NCßcatenin in transgenic mouse skin. Transgenic skin from a neonatal transgenic and control WT mouse was frozen in liquid nitrogen and ground to a powder. After extraction, proteins were resolved by electrophoresis through 8% polyacrylamide gels and subjected to immunoblot analysis using an anti-HA and anti–ß-tubulin (loading control) antibody and ECL chemiluminescence. The same experiment was performed on protein extract isolated from primary keratinocytes derived from neonatal WT and transgenic mouse skin. For protocol for isolation of primary keratinocytes, and making protein extracts, see Materials and methods. (B) Immunostaining on frozen sections of neonatal tail skin (left) and head skin (right) with anti-HA antibody, depicting expression of the transgene in basal epidermal cells and the follicle ORS. Some staining is also observed in the stratified differentiated layers of NCßcatenin expressing transgenic skin, perhaps due to the stability of the transgenic product. (C) The anatomy of the mature hair follicle. The mature follicle consists of an ORS contiguous with the basal layer of the epidermis (green cell layer), an inner root sheath that serves as the channel from which the hair exits the skin surface (pink), and the hair shaft itself (red). The hair is derived from the precortical cells, which along with the inner root sheath are descendents of proliferating matrix cells at the bulb of the follicle. Matrix cells are thought to maintain their proliferative state through sustained association with dermal papilla (yellow), encloaked by the matrix.

NCßcatenin-expressing transgenic mice display some features resembling a loss-of-function phenotype: switch from a hair cell to epidermal cell fate

View larger version (142K): [in this window] [in a new window]   Figure 3. Formation of epidermal cysts in NCßcatenin mice, at the start of postnatal hair cycle. (A–C and D–G) Hair cycle in WT and transgenic skin, respectively. (D–H) Progression of hair cycle and cyst formation in the skin of transgenic mice. Skin sections from day 24–28 NCßcatenin transgenic line and control littermates (WT) were fixed in 4% PFA fixative, embedded in OCT compound and sectioned (10 µm), and stained with hematoxylin and eosin and in some cases with Oil Red O to mark cells of sebaceous lineage. Shown are brightfield images of representative sections of: (A) onset of anagen; (B) formation of secondary hair germ; and (C) formation of new hair matrix in WT tail follicles. (D) NCßcat transgenic skin at the onset of anagen; (E) transgenic follicle displaying the formation of a small cyst-like structure; (F) early anagen transgenic follicle depicting the rapid formation of cyst, with Oil Red O staining in red sebaceous; (G) 2-mo-old skin follicle displaying late stage large cysts emanating from an old follicular structure. Higher magnification in inset shows epithelial invaginations emanating from the upper ORS.

The outermost layer of cells surrounding the follicular cysts exhibited morphological features typical of the basal layer of the epidermis (Fig. 4, compare A, epidermis, with B, cyst). Progressively more inward, the cyst resembled terminally differentiating epidermis. Large suprabasal-like cells were seen within the range between the outer basal-like layer and the middle of the cyst, whereas cells packed with keratohyalin granules were seen further inward. Enucleated squamous-like cells were found at the cyst center. Immunofluorescence with antibodies against a variety of epidermal differentiation markers confirmed the epidermal character of the cysts (Fig. 4, C and D). The basal-like cells at the periphery of the cyst were positive for K5 and K14, the suprabasal cells stained with antibodies against the spinous layer marker keratin 1 (K1), and the granular cells labeled with antibodies against filaggrin and loricrin, typically seen in the epidermal granular layer.

View larger version (77K): [in this window] [in a new window]   Figure 4. Epitheliod cysts in NCßcatenin mice display epidermal characteristics and lose the expression of hair follicle markers. 2-mo-old tail skin from NCßcatenin mice was harvested for H and E staining and immunofluorescence on the epitheliod cysts, formed in transgenic animals. The tissue was embedded in OCT, cut into 10-µm sections, and processed for staining purposes. Shown are bright field images of representative examples of (A) 40x magnification of H and E staining of transgenic interfollicular epidermis; and (B) the periphery of a cyst showing epidermal characteristics such as basal cells (arrows) and the supra-basal layers (straight bracket). (C and D) Confocal images of immunostaining for early and late differentiation markers, respectively. K1 and filaggrin are red and basal marker K5 is depicted in green. Filaggrin is not always expressed all around the epitheliod cyst. (E–K) Immunostaining for hair follicle markers in WT and transgenic skin follicles. (E) TCF3 staining in ORS of WT hair follicle in red. (F) Loss of TCF3 in the basal layer of the epidermal cysts. (G) remnants of some TCF3 positive cells in an early cyst, suggesting TCF3 expression is lost or fails to be maintained in the basal-like cells as the follicles develop into epitheliod cysts. (H) WT hair follicles expressing AE15, inner root sheath marker in blue, and LEF1 in the precortex in green. (J) AE13, pan-hair keratin marker in green, and LEF1 in red. (K) Representative example of loss of LEF1 (in red) and AE13 (in green) hair follicle markers in the epitheliod cysts formed in NC transgenic mice. Note that the asterisks mark background, noncellular staining of mouse monoclonal antibodies on scar tissue found in transgenic skin. precx, precortex.

Overall, the marked morphological and biochemical resemblance of these cysts to masses of differentiating epidermal cells paralleled the description of the cysts seen when ß-catenin was conditionally ablated in K14-expressing cells of mice (Huelsken et al., 2001) and when transgenic mice were engineered to express a K14-NLef1 transgene, encoding a form of Lef1 unable to associate with ß-catenin (Merrill et al., 2001; Niemann et al., 2002). In contrast to the striking effects on the hair cycle, the skin epidermis of K14-NCßcatenin mice was only modestly affected (Fig. 4 A). The patterns of K5, K1, and filaggrin expression in transgenic epidermis appeared largely normal (unpublished data), although the spinous layer and granular layers were somewhat thickened in some areas. The increased thickening was attributed to enhanced proliferation, as judged by staining with an antibody against Ki67, present in the nuclei of proliferating basal epidermal cells (unpublished data). These changes aside, the overall spatial and temporal pattern of epidermal differentiation was largely similar between WT and K14-NCßcatenin transgenic skin, and in some areas, no major differences were noted (Fig. 3, D and E).

Expression of NCßcatenin generates a failure to initiate Lef1/Tcf/ßcatenin-mediated signaling in the bulge
Given the similarities between the epidermal cysts of NCßcatenin and ß-catenin–null skin, and the inhibitory effects of NCßcatenin on Lef1/Tcf/ß-catenin–regulated gene transcription in vitro, it seemed most likely that the cysts arose from a failure to activate this transcription pathway. To test this, we mated our previously generated TOPGAL transgenic mice on the background of the NCßcatenin transgenic mice, and examined the double transgenic offspring for their ability to transactivate TOPGAL. In normal TOPGAL mice, a few cells within the bulge of many hair follicles expressed ß-galactosidase at the start of the first postnatal hair cycle, when the mesenchymal dermal papilla was abutted against the epithelial stem cell compartment (Fig. 5 A; DasGupta and Fuchs, 1999). When bred against the background of our previously generated K14-Nßcatenin transgenic mice, very strong TOPGAL activity was observed in most bulge cells undergoing hair cycle initiation (Fig. 5 B). In striking contrast, the bulges at the equivalent stage of NCßcatenin transgenic follicles were usually devoid of ß-galactosidase activity (Fig. 5 C). The finding that the cells in the bulge compartment of NCßcatenin expressing follicles were silent for TOPGAL activity and resulted in epidermal cyst formation provided compelling evidence that a loss of Tcf/Lef1/ß-catenin–mediated signaling activity in the bulge leads or contributes to the acquisition of epidermal cell fates.

View larger version (106K): [in this window] [in a new window]   Figure 5. Failure of TOPGAL activation in the bulge of NCßcatenin transgenic follicles, at the onset of the first anagen. Mouse skin was harvested at the start of anagen, embedded in O>C>T compound and snap frozen for later use. Shown are representative examples of 10-µm sections that were fixed for 2 min in 0.1% glutaraldehyde and subsequently stained with X-gal staining solution. (A) TOPGAL activation of one cell (arrowhead) in the bulge region of WT anagen follicle. (B) Superactivation of TOPGAL in cells streaming out of the bulge in K14-N87ßcat transgenic mice. (C) Lack of TOPGAL activity in the bulge of NCßcat transgenic follicles at the start of the new hair cycle.

NCßcatenin-expressing transgenic mice display some features resembling a gain of function phenotype: switch from an epidermal cell to a hair cell fate

View larger version (115K): [in this window] [in a new window]   Figure 6. NCßcat transgenic mice display gain-of-function ß-catenin signaling phenotype in interfollicular epidermis. Tail skin sections from day 28 NCßcatenin transgenic line and control littermates (WT) were fixed in 4% PFA fixative, embedded in OCT compound and sectioned (10 µm), and stained with hematoxylin and eosin. Shown are representative sections of A and B, tail skin follicles of WT and transgenic littermates, respectively. Hematoxylin and eosin staining revealed a phenotype in the interfollicular epidermis, reminiscent of that obtained in the Nß-catenin transgenic mice. (C) X-gal staining in transgenic mice harboring both NCßcatenin and TOPGAL. TOPGAL is activated in the de novo hair-like invaginations. (D) Multiple epithelial invaginations sprouting from the upper ORS of older follicles and the interfollicular epidermis of transgenic skin. (E) Confocal immunofluorescence image of WT 28-d tail skin, with Lef1 in green and ß-catenin in red. The epidermis displays very low levels, if any, of Lef1 staining (compare staining intensity with the nuclear Lef1 in precortex). (F and G) 28-d NCßcatenin transgenic tail skin stained for Lef1 in red and K5 in green. Arrowheads denote activation of Lef1 in hair placodes and hair germs. (F and G) Arrows denote ectopic expression of LEF1 in the basal layer of epidermis of 28-d transgenic animals.

Overall, the effects of NCßcatenin on epidermal cells was as a positive stimulator of ß-catenin signaling, thereby activating the hair cell fate and eventually leading to hair cell tumors, or pilomatricomas in all transgenic mice. This was in marked contrast to the transgene's effects as an inhibitor of ß-catenin signaling in the follicle precursor cells, where the outcome was epidermal cyst formation. The opposing effects were at least in part due to differences in transcriptional activation of Lef/Tcf/ß-catenin–regulated genes, as revealed by the fact that the epidermal cysts at sites anticipated for hair follicles were TOPGAL negative, whereas the follicle-like structures at sites anticipated for epidermis were TOPGAL positive. Most remarkably, the opposing effects of the transgene were not random, but rather were displayed in a highly cell type–specific fashion.

NCßcatenin is able to interact with E-cadherin and with members of the ß-catenin degradation machinery
Previous studies have shown that NCßcatenin can associate with Lef/Tcf family members, and our in vitro studies show that it can influence transactivation potential of Lef-regulated genes in keratinocytes. To assess how NCßcatenin is capable of having positive effects on epidermal cells at the same time as it has negative effects on hair cells, it was necessary to confirm that this protein is also able to associate with candidate cytoplasmic partners, including the adherens junction protein E-cadherin and APC, the protein that targets ß-catenin for the proteosome degradation pathway.

Cell lysates from skin and calcium-treated primary keratinocytes and from skin of WT and transgenic mice were first adjusted to have equal levels of E-cadherin in the starting lysate extracts for WT and transgenic (Fig. 7). Anti–E-cadherin antibody was then used in a pulldown assay to bind E-cadherin complexes to protein G–conjugated Sepharose beads. These complexes contained both HA-tagged NCßcatenin and endogenous ß-catenin (Fig. 7). In skin and in primary keratinocytes, the overall E-cadherin pulled down in the assays was consistently higher in the transgenic versus the WT samples, despite the roughly comparable levels in the aliquots used for pulldown.

View larger version (45K): [in this window] [in a new window]   Figure 7. Biochemical interactions of NCßcatenin with E-cadherin and APC. (Top) Interactions of NCßcatenin with E-cadherin. Coimmunoprecipitation studies were performed to assay interaction between NCßcatenin and E-cadherin. Total protein extracts were made from WT and NCßcat transgenic skin and primary keratinocytes by solubilizing in strong lysis (RIPA) buffer. Shown on the right panel are results from Western blot analysis of -E-cadherin immunoprecipitates, probed with anti-E-cadherin, anti-HA, and anti–ß-catenin antibodies. On the left panel, the same antibodies were used to detect total protein levels, before the pulldown (input) with anti–E-cad antibody. The anti-E-cad antibody could successfully pull down E-cadherin and the proteins associated with it. Extracts to which anti–E-cad antibody was not added, failed to pull down the complex (lane 5, right panel). (Bottom) Interactions of NCßcatenin with APC. Experiment was performed as in top, except that anti-APC antibody was used in place of anti–E-cad.

The ratio of nuclear NCßcatenin to endogenous ß-catenin is lower in epidermal than in hair cells, and yet K14 promoter activity is higher in epidermal than in hair cells
Without a COOH-terminal activation domain, ß-catenin is unable to function with Lef1/Tcf family members to transactivate genes. To understand how NCßcatenin might be eliciting positive effects on interfollicular and upper ORS epithelium, we first examined the location of NCßcatenin when transiently expressed in keratinocytes. To distinguish transgenic from endogenous ß-catenin, we used an anti-HA antibody to recognize the epitope tag of the transgene product and an anti–ß-catenin antibody directed against the COOH terminus of ß-catenin to specifically recognize the endogenous ß-catenin protein by immunofluorescence. The results are summarized in Fig. 8. Whereas only 2–5% of primary keratinocytes were transfected, 40% of them exhibited nuclear as well as cytoplasmic localization of the transgene product (Fig. 8 A, NCßcat in green). Interestingly, 100% of transfected cells exhibited nuclear localization of endogenous ß-catenin (Fig. 8, A and B, endogenous ß-catenin in red). Under the low-calcium conditions used here, endogenous ß-catenin in untransfected and transfected cells was always cytoplasmic. However, in stark contrast to the nuclei of transfected cells, the nuclei of untransfected cells were negative for endogenous ß-catenin. These findings suggest that by expressing NCßcatenin protein in keratinocytes, endogenous ß-catenin is translocated to the nucleus, a point which we confirmed by immunoblot analyses (unpublished data). Moreover, the data demonstrate at a molecular level that the transgene product can displace endogeous ß-catenin from the cytoplasmic pool, and stimulate a process that results in its accumulation in the nucleus. The generation of nuclear ß-catenin in transfected cells is likely to explain why our transfections with NCßcatenin always gave a slight enhancement of TOPGAL activity when coexpressed with Lef1 (Fig. 1 C).

View larger version (51K): [in this window] [in a new window]   Figure 8. ß-Catenin translocates into the nucleus of cells transfected with NCßcatenin. Shown are representative samples of immunofluorescence (confocal) images of mouse keratinocytes transfected with K14-NCßcatenin (A and B) and of primary keratinocytes derived from NCßcatenin–expressing transgenic mice (C and C'). Endogenous ß-catenin staining is shown in red; and anti-HA staining in green. The anti-HA antibody detects the HA-tagged transgenic protein product whereas the anti–ß-catenin antibody specifically detects the endogenous ß-catenin, as it is directed toward the COOH terminus of the protein which is absent from the NCßcatenin transgenic product. In mouse keratinocyte cell line, endogenous ß-catenin translocated to the nucleus, only in cells transfected with K14-NCßcatenin. The same phenomenon was seen in primary keratinocytes, derived from the K14-NCßcatenin transgenic mice. (D–G) Increase in cytosolic and nuclear ß-catenin in NCßcatenin transgenic mice. Shown are confocal images of immunostaining for endogenous ß-catenin in red and anti-HA in green. (D and E) Day 28 WT tail epidermis and hair bulb, respectively. Note the absence of cytoplasmic (and the predominantly membrane) staining for endogenous ß-catenin in the WT epidermis (D). Also note nuclear ß-catenin (arrowheads) staining in the precortical cells of the hair bulb (E). (F and G) Age-matched sections of transgenic epidermis and epithelial invaginations in NCßcatenin transgenic skin. Note increased cytoplasmic ß-catenin in transgenic epidermis (F, arrowheads) and nuclear ß-catenin in hair-like invaginations (G, arrows; G' depicts a high magnification view of G, arrowheads).

In WT epidermis in vivo, ß-catenin is normally restricted to cell–cell borders, and is not found in the cytoplasm or nucleus (Fig. 8 D). However, in the hair follicle, cytoplasmic and nuclear ß-catenin are readily detected in the hair progenitor cells, referred to as precortex (Fig. 8 E). The precortex (Fig. 8, arrowheads) is where activation of Lef1/ß-catenin regulated genes, including the hair-specific keratins and TOPGAL, typically occurs (DasGupta and Fuchs, 1999; Merrill et al., 2001).

The pattern of endogenous ß-catenin was dramatically different in NCßcatenin epidermis (Fig. 8 F). In addition to cell–cell border staining, which was seen with antibodies against both the HA-tagged NCßcatenin gene product and endogenous ß-catenin, cytoplasmic staining was also prominent, particularly for endogenous ß-catenin (Fig. 8 F). In the TOPGAL-positive, flower-like downgrowths of NCßcatenin skin, nuclear ß-catenin was also detected (Fig. 8 G, arrowheads). In these downgrowths, the ratio of FITC (transgene product) to Texas red (endogenous ß-catenin) was always higher at cell borders than in the nuclei (Fig. 8 G', inset, higher magnification). The preferential detection of endogenous nuclear ß-catenin was consistent with the presence of Lef1 and TOPGAL activity in these downgrowths.

Interestingly, in the early stages of epidermal cyst formation, we occasionally captured follicle-like structures where intense anti-HA staining, reflective of NCßcatenin, was detected in the nuclei of the precortex (Fig. 9 E). At these early stages, HA nuclear staining was sometimes also seen in the bulge compartment (Fig. 9 F). These findings are consistent with the dominant negative action of NCßcatenin in transcriptional regulation and with our failure to detect TOPGAL activity in the bulge or the precortex of the NCßcatenin follicles. In epidermal cysts, peripheral basal-like cells showed intense border staining for the K14 promoter-driven transgene product, paralleling the intensity seen in the K14-positive epidermal basal layer and the ORS (Fig. 9 D, arrows). In these regions, some nuclei stained with HA antibodies (Fig. 9 D'', inset, arrows; higher magnification of such HA-positive nuclei). The more central, spinous-like cells did not display nuclear HA staining, and in fact expressed considerably less NCßcatenin, consistent with the switch from K5/K14 to K1/K10 and the downregulation of K14 promoter activity in these layers. These results provide additional evidence that expression of NCßcatenin in the basal-like cells of developing cysts is high and able to block the transactivating functions of ß-catenin.

View larger version (75K): [in this window] [in a new window]   Figure 9. Plasticity of the epitheliod cysts formed in NC transgenic skin. Differences in the levels of NCßcat and endogenous ß-catenin could influence cell fate decisions in the epitheliod cysts. (A). Hematoxylin and eosin combined with X-gal staining on epitheliod cysts formed in mice harboring both NCßcatenin and TOPGAL. A few cysts display X-gal staining which coincides with activation of Lef1 (in red) and hair keratins, as judged by AE13 (pan-hair keratin antibody) staining in green in a similar section (B and C, confocal image). All sections were 10 µm in thickness and fixed in 4% PFA. Sections processed for X-gal staining were fixed in 0.1% glutaraldehyde. (C–F) Levels of endogenous ß-catenin and NCßcatenin complexes with Lef1 determines epidermal versus hair cell fates in epitheliod cysts. (C) Immunostaining for Lef1 (in red) and AE13 (in green) D demonstrate high levels of nuclear Lef1 (white arrowheads) in both NCßcatenin–expressing basal cells and high ß-catenin–expressing suprabasal cells. Moreover, cells with Lef1 and higher levels of endogenous ß-catenin display AE13-positive staining (hair cortex marker), whereas the basal cells are not immunoreactive toward AE13 antibody. (D) Confocal immunofluorescence image of a cyst stained for endogenous ß-catenin (in red) and the epitope HA (in green). Insets D' and D'' display diffuse nuclear ßcat and nuclear HA staining, respectively. Note the reciprocal levels of staining between the HA-tagged transgenic NCßcat protein and endogenous ß-catenin in the epitheliod cysts. (D) Antibody against HA-tagged NCßcatenin protein (in green) detected the transgene product in the basal cells of the cysts. However, staining for endogenous ß-catenin (in red) showed lower levels in basal cells but higher staining in suprabasal cells that have much lower levels of NCßcatenin. (E and F) Confocal images of early stages of cyst formation with E, an occasional precortex like structure with high levels of nuclear NCßcat (as seen by HA antibody staining in green, arrowheads) and (F) diffuse nuclear HA staining in the bulge (Bu) of NC transgenic follicle.

Interestingly, despite considerable ß-catenin in the cytoplasm of spinous epidermal cells (e.g., Fig. 8 F), AE13 and TOPGAL staining was not seen in differentiating epidermis. Thus, although the epidermal cysts appeared morphologically and biochemically similar to epidermis, they retained a certain level of atypical plasticity, and were able to revert midstream from an epidermal to a hair program of terminal differentiation.

To summarize the data presented in Figs. 8 and 9, there were marked differences in the relative levels of NCßcatenin and endogenous ß-catenin in the nuclei of different epithelial cells within the skin of the transgenic mice. The ratio of nuclear NCßcatenin to endogenous ß-catenin seemed to be highest in hair follicle cells and lowest in epidermal basal cells and upper ORS cells. These differences correlated well with whether the transgene acted in a negative or positive fashion to influence TOPGAL activity. These differences also correlated well with the type of epithelial cell within the skin and its relative stage of differentiation along a particular lineage. The differences did not correlate with K14 promoter activity.

	Discussion

Top Abstract Introduction Results Discussion Materials and methods References

 
Molecular insights into NCßcatenin's loss of function phenotypes in the stem cells and hair lineage of the skin
Although NCßcatenin has the potential to exert both activating and repressor effects (Miller and Moon, 1997; Cox et al., 1999), the protein behaved primarily as a repressor in mouse keratinocytes in vitro. Anticipating that NCßcatenin's actions in skin keratinocytes in vivo would parallel those in vitro, we expected to see an abrogation of ß-catenin–mediated nuclear signaling and a maintenance of adherens junctions in transgenic skin epithelium. In agreement with our expectations, the epidermal cysts that formed at the start of the first postnatal hair cycle were similar to those described by Huelsken et al. (2001), who used K14-Cre recombinase to conditionally ablate ß-catenin gene expression in skin.

In ß-catenin–null skin, plakoglobin is able to associate with E-cadherin to assemble adherens junctions (Aberle et al., 1996; Huber et al., 1997). In NCßcatenin skin, NCßcatenin and endogenous ß-catenin both localized to adherens junctions. Despite these differences in armadillo proteins at cell–cell junctions, the ß-catenin–null and NCßcatenin mice displayed similar epidermal cysts, suggesting that the cysts arose from a failure to activate ß-catenin/Lef1/Tcf–regulated downstream target genes, rather than changes in intercellular junctions per se. Consistent with this notion is our finding that TOPGAL activation was largely silent during the postnatal hair cycles that led to epidermal cyst formation at the expense of hair differentiation. These findings were in good agreement with, and extended those of, Huelsken et al. (2001) and Niemann et al. (2002).

Our in vitro and in vivo assays tell us that elevated levels of Nßcatenin, but not NCßcatenin, can activate TOPGAL in the stem cell compartment of the hair follicle. The failure to transactivate ß-catenin/Lef/Tcf–regulated genes in the bulge of K14-NCßcatenin mice did not appear to affect the downgrowth of the secondary hair germs. However, the process of their subsequent development into hair follicle was severely perturbed.

The nuclear HA staining and reduced endogenous ß-catenin staining of the precortical cells of the K14-NCßcatenin secondary hair germs provided an explanation for the dominant negative effects of the transgene on hair cells. However, the strong HA staining could not be explained by K14 promoter activity, which is actually very low in matrix and precortical cells (Byrne et al., 1994; Wang et al., 1997). Moreover, the strong nuclear HA staining could not be explained by a simple canonical Wnt signaling mechanism, which should have increased nuclear levels of endogenous ß-catenin without affecting levels of NCßcatenin. Rather, the preferential concentration of nuclear NCßcatenin over endogenous ß-catenin seemed to be a reflection of a novel mechanism that either specifically stabilizes ß-catenin through its armadillo repeats or preferentially translocates the truncated ß-catenin to the nucleus. Although the molecular nature of the underlying mechanism remains to be explored, its existence is one that seemed to occur preferentially in the bulge, matrix and precortex, i.e., cells known to express members of the Tcf/Lef1 family of HMG proteins.

Molecular insights into NCßcatenin's gain of function phenotypes in the epidermis and upper ORS of the skin
An important and revealing feature of our NCßcatenin transgenic mice was the surprising existence of a gain-of-function phenotype within the interfollicular and upper ORS epithelium of the skin. By all criteria examined, these NCßcatenin-expressing cells behaved analogously to the equivalent cells of the Nßcatenin mice. Thus, whereas NCßcatenin elicited a dominant negative effect on keratinocytes in vitro and on skin epithelial stem cells and follicle cells in vivo, it elicited a positive effect on interfollicular and upper ORS epithelium. The interfollicular and upper ORS epithelium are thought to be derived from the stem cells in the bulge. How can we account for the remarkable difference between stem cells and their progeny in the way they respond to this single transgene protein?

Several key observations help us to understand this paradox. One important clue is that despite the inability of NCßcatenin/Lef/Tcf complexes to transactivate Lef1/Tcf regulated genes on their own, TOPGAL was ectopically activated in the epidermis and the ORS at sites of hair germ like invaginations. Conversely, as detected through TOPGAL activity, normal Lef/Tcf/ß-catenin regulated gene activity was impaired in the bulge and precortical cells of postnatal transgenic follicles. Because the transgene protein lacks a transactivation domain, its positive actions on TOPGAL activity must then be indirect. In this regard, our in vitro studies revealed endogenous ß-catenin in the nuclei of NCßcatenin–transfected cells, and in vivo, nuclear endogenous ß-catenin was seen in areas where TOPGAL was activated.

How then does NCßcatenin lead to generation of critical threshold levels of stabilized ß-catenin in interfollicular epidermal cells and in the upper ORS, but not in the bulge or in the precortex? Our analyses left us to conclude that it is a property inherent to the character of each epithelial skin cell type that determines how the cell will respond to NCßcatenin. Thus, ironically, hair follicle–like invaginations were restricted to transgenic upper ORS and epidermis, whereas epidermal transdifferentiation was restricted to transgenic cells that normally would adopt a hair cell fate.

At sites of epithelial downgrowth in the interfollicular epidermis or upper ORS, the pools of cytoplasmic and nuclear endogenous ß-catenin seemed to increase. One way in which this might be achieved is that in epidermis, NCßcatenin localizes strongly to cell–cell junctions, where it may displace endogenous ß-catenin and raise its cytoplasmic pool. Localization of the transgene protein to cell–cell junctions sequesters it, physically restraining it from interfering in the nucleus. Intriguingly, cell–cell junctions are fewer in hair follicle precursor cells (Nanba et al., 2000), perhaps increasing the likelihood that NCßcatenin will enter the nuclei of these cells and act to impair Lef1/Tcf gene activity.

Another contributing factor is likely to be NCßcatenin's association with APC (for review see Bienz, 1999; Mimori-Kiyosue and Tsukita, 2001). Through NCßcatenin-mediated sequestering of APC or other members of the ß-catenin degradation machinery, endogenous ß-catenin could be stabilized. The preferential concentration of endogenous ß-catenin in the nuclei of cells at epithelial downgrowths suggests that NCßcatenin may preferentially associate with either or both of these interacting partners.

Our transient transfection experiments with cultured keratinocytes demonstrate that even under conditions where cell–cell junctions do not form, endogenous ßcatenin from the cytoplasm can translocate to the nucleus when cells express the transgene. This finding is compatible with a displacement mechanism that involves NCßcatenin-mediated sequestration of either a component of the proteosome degradation machinery or a component that keeps endogenous ß-catenin from entering the nucleus. APC has been implicated in both processes, and its ability to associate with transgenic and endogenous ß-catenin makes it a good candidate for explaining these observations. Overall, our in vitro and in vivo observations corroborated similar studies in Xenopus (Miller and Moon, 1997) and in Drosophila (Cox et al., 1999), where positive actions of a membrane-tethered form of ß-catenin also prompted investigators to posit that competition for E-cadherin and APC may displace endogenous ß-catenin into the nucleus. A related parallel stems from altering E-cadherin levels, which also results in phenotypes consistent with sequestering signaling-competent ß-catenin away from the nucleus (Orsulic et al., 1999; Ciruna and Rossant, 2001).

Another intriguing phenomenon that is likely relevant to our understanding of Lef1/Tcf/ß-catenin action in skin epithelium is the elevated Lef1 mRNA and protein expression which occurs in epithelial invaginations transgenic for either Nßcatenin (Gat et al., 1998) or NCßcatenin (present study). This feature suggests the existence of a positive feedback mechanism that could lead to Lef1 transcription when ß-catenin levels are stabilized in epidermis. Because the Lef1 promoter contains functional Lef1/Tcf binding sites (Hovanes et al., 2001 and references therein), it is possible that displacement of endogenous ß-catenin frees it to associate with small amounts of Lef1 which could then initiate this cycle of upregulated Lef1 gene expression. Further experiments will be necessary to fully understand this mechanism. In addition, recent studies on E-cadherin have also provided new insights into how altering the levels of adherens junction proteins may modulate Wnt-mediated signaling in morphogenetic processes (Orsulic et al., 1999; Ciruna and Rossant, 2001).

A number of other ß-catenin interacting proteins have been identified in recent years, and a priori, some of these proteins may also be involved in accounting for the unexpected transactivating-like phenotype associated with NCßcatenin in skin. Consistent with this notion are transcriptional repressor proteins such as XSox17, XSox3, and the Smad proteins, which have been shown to physically interact with ß-catenin (Zorn et al., 1999; Schohl and Fagotto, 2002), and in this regard, the transgene protein might act to titrate out some of these repressors. In contrast, the repressor proteins reptin52 and pontin52 have recently been identified as COOH terminally interacting ß-catenin–associated repressor proteins, which would render them insensitive to the transgene protein (Bauer et al., 2000; Etard et al., 2000; Takemaru and Moon, 2000). However, for these proteins to be involved, NCßcatenin would have to function by relief of ß-catenin/Tcf-mediated transcriptional repression, a mechanism previously invoked to explain certain positive phenotypes obtained with the NCßcatenin mutant (Funayama et al., 1995; Miller and Moon, 1997). This seems unlikely for epidermis because: (a) the nuclear ß-catenin seen in the activated cells was mostly endogenous rather than transgene product; (b) the epidermis and upper ORS do not normally express appreciable levels of Lef1/Tcf family members; and (c) the Lef/Tcf member activated at these sites was Lef1, acting primarily as a coactivator and not a repressor. However, repression might still be relieved if endogenous nuclear ß-catenin competes for the binding of Tcf/Lef repressor proteins, e.g., Grg/Tle (Merrill et al., 2001).

Reconciling two opposing actions from the same transgene
Ironically, by expressing NCßcatenin, the epidermal-like cells ended up generating hair follicle–like phenotypes, whereas the hair follicle–like cells ended up generating epidermal-like phenotypes. These findings imply that the behavior of NCßcatenin in epithelial cells of the skin is highly tailored to the particular stem cell lineage and stage of differentiation of the skin cell. The final outcome of epithelial skin cell fate then appears to be dictated not only by the levels and location of ß-catenin in a cell, but also the relative levels of other ß-catenin interacting factors in the cell. An interesting point that supports this conclusion is that by downregulation of transgene expression in differentiating epidermal cysts, the former hair cells turned epidermal cells reverted back to their hair cell fate, activating hair keratin gene expression. Such reversibility of cell fates was not observed in the bona fide epidermis, indicating that suppression of nuclear ß-catenin in a hair precursor cell required sustained transgene expression to be maintained, whereas epidermal precursor cells have some intrinsic mechanism to prevent this from happenin